The long-term goal of our currently funded work is to determine if the coreceptor-binding site, or the smaller sub-region participating in sulfotyrosine association, is a useful epitope for candidate vaccines or a practical target of therapeutics. The goal of this current proposal is to determine if broadly neutralizing and highly potent coreceptor-binding site antibodies can be generated. The existence of such antibodies would motivate efforts by our own lab and others to elicit these antibodies in vivo. Study of these coreceptor-binding site antibodies will also highlight properties contributing to their breadth and potency. [unreadable] Sulfotyrosines are necessary for an antibody to closely mimic CCR5. Also, due to their biophysical properties, sulfotyrosines can make substantial contributions to the binding energy of protein-protein interactions. Finally, as we show here, the sulfate-binding region of gp120 is exposed in the absence of CD4, perhaps because it is also a site for low-affinity proteoglycan association. It is therefore likely that most broadly and potently neutralizing coreceptor-binding site antibodies will include sulfotyrosines in their antigen-binding site. Unfortunately, conventional phage-based selection does not permit selection of tyrosine-sulfated peptides or proteins. We will therefore apply two novel and complementary approaches to the improvement of existing coreceptor-binding site antibodies, and, later, to the generation of new antibodies of this class. The first approach uses conventional phage based selection, except that phage bearing single-chain antibody are generated in a novel bacterial system capable of expressing tyrosine-sulfated proteins. This system, recently developed in the laboratory of Peter Schultz, uses a novel tRNA/tRNA synthetase pair to incorporate sulfotyrosines at positions encoded by amber codons. The second approach uses stable-isotope labeling and high-mass-accuracy mass spectrometry to identify high-affinity scFv from a narrower panel of scFv variants. Although the starting diversity is necessarily more limited than with phage-based selection, this approach imposes fewer constraints on the number of sulfotyrosines that can be introduced into an scFv. [unreadable] Our aims for this revision proposal are: (1) to develop these two technologies, and (2) to use them in parallel to improve the breadth and potency of 412d and E51, the two most potent of the described tyrosine-sulfated antibodies. These studies will complement our current efforts to evaluate the utility of the coreceptor-binding site as a target for HIV-1 vaccines, and develop two novel technologies that will be useful to the future study of HIV-1 neutralizing antibodies. [unreadable] [unreadable] [unreadable]